Large Variabilities in Host Strain Susceptibility and Phage Host ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2007, p. 6730–6739 Vol. 73, No. 21
0099-2240/07/$08.000 doi:10.1128/AEM.01399-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Large Variabilities in Host Strain Susceptibility and Phage Host Range
Govern Interactions between Lytic Marine Phages and Their
Flavobacterium Hosts †
Karin Holmfeldt, 1 Mathias Middelboe, 2 Ole Nybroe, 3 and Lasse Riemann 1 *
Department of Natural Sciences, Kalmar University, S-391 82 Kalmar, Sweden 1 ; Marine Biological Laboratory,
University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingør, Denmark 2 ; and Department of Ecology,
University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 3
Received 25 June 2007/Accepted 23 August 2007
Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial
community composition through host-specific infection and lysis. In the present study we demonstrate for a
marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and
lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three
Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic
and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae).
Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6
orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced
variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate
that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards
phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an
extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community
composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies
based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.
Marine viruses are the most abundant biological entity in the
world’s oceans (4) and are a main mortality factor for marine
bacterioplankton. Most marine virus particles are believed to infect
bacteria, and it has been estimated that 10 to 20% of planktonic
marine bacteria are lysed by viruses (phages) per day (55).
Thereby, up to a quarter of the photosynthetically fixed carbon in
the ocean is recycled back to dissolved organic material (65).
Phage infection affects bacterial evolution by transferring genetic
material between bacteria (41, 62). Further, by selective
infection and lysis of predominant bacterial populations, it is
thought that lytic phages control bacterial community composition
and sustain coexistence of bacterial species (“killing the
winner” [57, 58]). This view is supported by predator-prey-like
oscillations of phage-host systems in aquatic environments (19,
69) but is likely to simplify actual conditions, as it does not
account for broad-host-range phages or bacterial acquisition of
resistance (45).
Many marine phages appear to have a narrow host range,
infecting only the bacterial strain with which they were isolated
(24, 38, 40, 63). However, some findings indicate that the
dogma of extreme host specificity among bacteriophages may
be an artifact produced by commonly used methods for phage
isolation. For instance, the single-host enrichment protocol
may select for phages with a narrow host range (22, 64), and
* Corresponding author. Mailing address: Department of Natural
Sciences, Kalmar University, S-391 82 Kalmar, Sweden. Phone: 46 480
447334. Fax: 46 480 447340. E-mail: Lasse.Riemann@hik.se.
† Supplemental material for this article may be found at http://aem
.asm.org/.
Published ahead of print on 31 August 2007.
infection and lysis by low-virulence phages, which typically
have a broad host range (22), may not be detected in plaque
assays (10). However, some marine phages are known to infect
different strains of the same species (12, 51, 64) or even different
species (10–12). Hence, the specificity of phage-host
interactions in marine waters needs to be better characterized
and understood.
Development of resistance is a common bacterial response
to the presence of lytic phages. Chemostat experiments have
shown that when exposed to lytic phages, the composition of
heterotrophic marine bacterial populations (34, 35) and Escherichia
coli populations (13, 20, 30, 37) rapidly shifts from
dominance by phage-sensitive clones to dominance by phageresistant
clones. Consequently, phage-resistant bacterial mutants
seem to be constantly produced and tend to replace the
sensitive strains when the population is exposed to a strong
selective pressure from infectious phages. Further, for E. coli a
strong coevolution of resistant bacteria and phages with an
extended host range has been observed (27, 37). In a comprehensive
study of hundreds of phage-host systems from several
marine regions, a complex pattern of resistance and susceptibility
was observed (40). Similarly, Synechococcus communities
may consist of co-occurring resistant and sensitive strains (61).
Hence, acquisition and maintenance of resistance against
phage infection may be an integral part of marine bacterioplankton
ecology, but the incidence of resistance in natural
communities is difficult to evaluate directly (14).
In studies of bacterioplankton community composition, the
delineation of bacterial species has been based on the 16S
rRNA gene and, more recently, on the internal transcribed
spacer (ITS) (8, 47), providing the phylogenetic resolution
6730

VOL. 73, 2007 COMPLEXITY OF FLAVOBACTERIUM PHAGE-HOST INTERACTIONS 6731
governed by these particular gene regions. The limitations
associated with such species delineation has recently been discussed
in the context of strain-specific niche occupation (21),
where freshwater bacterial isolates with identical or almost-identical
16S rRNA gene and ITS sequences were suggested to represent
distinct populations and to probably occupy different ecological
niches (17, 21). These limitations may also apply in the
context of strain-specific phage-induced bacterial mortality.
In the present study a collection of 21 Cellulophaga baltica
(Bacteroidetes) strains, which were highly similar genotypically
and phenotypically, allowed us to address phage-host interactions
at the strain level. Our results demonstrate large strainspecific
differences in phage susceptibility among hosts and a
pronounced variability in specificity and efficiency of infection
for the C. baltica phages.
MATERIALS AND METHODS
Isolation of bacteria and phages. Strains NN014847 to NN016048 were isolated
from various Danish coastal waters in 1994 (23). Strains MM#3 and #4 to
#19 were isolated from Øresund surface water (56°2N, 12°37E) in February
2000, and strain OL12a was isolated from Baltic Sea surface water east off Öland
(56°37N, 16°45E) in April 2005. The strains were selected as yellow colonies on
CYT agar plates (1.0 g casein, 0.5 g yeast extract, 0.5 g CaCl 2 H 2 O, 0.5 g
MgSO 4 H 2 O, 15 g agar, 1,000 ml deionized water, pH 7.3) with colony morphology
(size, texture, and edge appearance) and restriction fragment length
polymorphism (RFLP) profiles (16S rRNA gene, HaeIII, and NdeII; Roche)
similar to those of the C. baltica type strain (strain NN015840) (23).
Phages were obtained from Øresund surface water in 2000 and 2005. After a
0.45-m prefiltration (Millipak; Millipore), phages were concentrated 250-fold
by tangential flow filtration (Millipore) followed by Amicon ultracentrifugation
(Millipore). Phages were isolated using the top-agar plating technique (plaque
assay) (48), with all of the isolated bacterial strains used as hosts. Plaques with
different morphologies were selected from each host and purified for three
rounds of infection and lysis. Thereafter, 5 ml phage buffer (0.1 M NaCl, 0.008
M MgSO 4 , 0.05 M Tris-HCl, 2 mM CaCl 2 , 0.1% gelatin, 0.5 M tryptophan, 5%
glycerol, pH 7.5) was added to a fully lysed plate, and the agar overlay was
shredded with a sterile inoculation loop. The plate was incubated on a shaker for
20 min, and the mixture was then transferred to a sterile tube and centrifuged
(5,000 g, 10 min). The supernatant was passed through a 0.22-m filter (Millex
GS; Millipore), and the phage stock was stored at 4°C.
In order to examine potential effects of resistance acquisition on host range,
strains resistant to two of the isolated phages, S M and S T , were developed. These
strains, #3 r S M and #3 r S T , were obtained after exposure of strain MM#3 to
the phages in laboratory experiments and isolated from fully lysed plates.
Sequencing of the 16S rRNA gene and the ITS. Bacterial DNA was extracted
using the DNeasy tissue kit (QIAGEN), and the 16S rRNA gene was PCR
amplified using PuReTaq Ready-To-Go Beads (Amersham Biosciences) and
primers 27f and 1492r (15). The thermal program consisted of 30 cycles of 30 s
of denaturation at 95°C, 30 s of annealing at 50°C, and 45 s of extension at 72°C,
followed by 7 min at 72°C. The ITS was amplified using primers G1 and 23Sr (21)
and a thermal program consisting of 35 cycles of 30 s of denaturation at 95°C, 30 s
of annealing at 54°C, and 45 s of extension at 72°C, followed by 72°C for 7 min.
DNA was sequenced bidirectionally (commercially by Macrogen, Korea) using
primers 27f, 530f, 519r, and 907r (26) for the 16S rRNA gene and primers G1 and
23Sr for the ITS. In some cases, the PCR-amplified ITS region could not be
sequenced directly. The PCR products were therefore cloned prior to sequencing
(TOPO TA cloning kit; Invitrogen). Sequences were aligned in
Seqman (DNASTAR), and phylogenetic trees were constructed using the
neighbor-joining algorithm in Clustal X (59).
Genotyping by UP-PCR. Universally primed PCR (UP-PCR) is a whole-genome
fingerprinting technique originally developed for fungi (see reference 31
for a review) and recently used to discriminate between genetically similar
bacteria (7, 70). The UP-PCR method uses a single primer to prime genomes
arbitrarily, and the primer is designed to primarily target less-conserved intergenic
areas (31). Crude DNA templates for PCR (cell lysates) were prepared
from each bacterial isolate as described by Brandt et al. (7) and stored at 20°C
until use. UP-PCR was performed using the universal L15/AS19 primer 5-GAG
GGT GGC GGC TAG-3 (32). PCR mixtures (20 l) consisted of molecular
biology reagent-grade water (Sigma), buffer (supplied with DNA polymerase), 2
mM MgCl 2 (supplied with DNA polymerase), 100 M of each deoxynucleoside
triphosphate (AH Diagnostics), 1 ng l 1 of PCR primer (DNA Technology), 1
unit of Dynazyme II DNA polymerase (Finnzymes, Finland), and 1 l template
DNA. PCR was carried out using the following program: initial denaturation at
94°C for 3 min and then 32 cycles consisting of denaturation at 94°C for 60 s,
primer annealing 53°C for 60 s, and elongation at 72°C for 60 s. In the first and
the last cycles, the elongation steps were extended to 90 s and 3 min, respectively.
PCR products were subjected to agarose gel electrophoresis (1.5%, 100 V, 40
min) and stained with ethidium bromide. A 100-bp DNA ladder (Fermenta) was
used as a molecular size marker. UP-PCR analysis was performed at least twice
for each isolate to verify that reproducible band patterns were obtained. Subsequently,
the isolates were grouped based on identical UP-PCR patterns.
Analysis of lipopolysaccharide (LPS) profiles. Bacterial strains were grown in
MLB medium (0.5 g Casamino Acids, 0.5 g peptone, 0.5 g yeast extract, 3 ml
glycerol, 500 ml filtered seawater [Øresund], and 500 ml demineralized water) at
room temperature for 72 h. Pseudomonas fluorescens strain Ag1, which was used
as a reference, was grown to early stationary phase in LB medium (10 g tryptone,
5 g yeast extract, 1 g glucose, 10 g NaCl, 1 liter Milli-Q) at 28°C.
For preparation of proteinase K-digested cell lysates, cells were centrifuged
(10,000 g, 5 min), washed once in phosphate-buffered saline, and resuspended
in lysis buffer (0.5 M Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate (SDS), 10%
glycerol). Cell concentrations were normalized during resuspension. Proteinase
K (75 g ml 1 ) was added and the lysates incubated at 56°C for 1 h, boiled for
5 min, centrifuged (14,000 g, 30 min), and stored at 20°C. Lysates were
analyzed by SDS-polyacrylamide gel electrophoresis using 14% acrylamide–0.4%
bisacrylamide and 4 M urea as described by Sørensen et al. (52), and a prestained
protein ladder (PageRuler Ladder Plus; Fermentas) was included on each gel.
After electrophoresis, gels were fixed in ethanol-acetic acid overnight and stained
with a periodic acid-silver stain (60). Specificity of the staining was ensured by
monitoring silver staining of LPS from the internal control (P. fluorescens Ag1)
without obtaining staining of the included protein ladder. The strains were
grouped based on similar LPS profiles. The profiles were reproducible between
independent replications, yet the intensities of individual bands, especially in the
low-molecular-weight region, varied between runs.
Specificity of phage infection. To determine phage host range and the bacterial
susceptibility to specific phages, a cross infectivity test where all bacterial strains
were exposed to all individual phage stocks was performed. Three microliters of
phage stock (10 8 to 10 11 PFU ml 1 ) was spotted onto a lawn of host bacteria in
top agar, and plates were examined for cell lysis after 1, 3, and 5 days. These spot
tests were performed three independent times. Infection was considered positive
when lysis (i.e., a clearing zone) was seen in all three tests. In spot tests, spots may
originate from inhibition of bacterial growth, often seen as turbid spots, rather
than direct viral lysis (39). To verify the presence of infectious phages, plaque
assays in dilution series were performed in some cases where turbid spots were
seen. In all cases plaques were obtained, suggesting that phage lysis caused the
clearing zones. An unweighted-pair group method using average linkages
(UPGMA) tree was constructed using the software Quantity One 4.2.1 (Bio-
Rad), where the lysis/no lysis matrix was converted to pairwise distances using
the Dice similarity coefficient.
Since several bacterial strains were susceptible to the same phage, we sought
to determine whether phage infection was equally efficient in different hosts.
Three hosts were exposed to the same phage titer, and infectivity was quantified
by plaque assay. PFU were examined after 1, 2, and 3 days. This was performed
for three different phages.
Phage genome sizes. Phage genome sizes were determined by pulsed-field gel
electrophoresis (PFGE). Freshly made phage stocks (see above) were concentrated
by ultracentrifugation (222,000 g, 2.5 h, 4°C; Beckman). DNA was
obtained according to the protocol for DNA (3) and quantified fluorometrically
(PicoGreen; Molecular Probes). Some phage genomes disintegrated when
extracted using this protocol. These were therefore lysed in agar plugs. Equal
amounts of phage concentrate and melted 1.5% low-melting-point agarose
(Sigma) in MSM buffer without gelatin (53) (450 mM NaCl, 50 mM MgSO 4 ,50
mM Tris, pH 8.0) were mixed, transferred to plug molds, and left to solidify at
4°C. The plugs were incubated in lysis solution (0.25 M EDTA [pH 8], 1% SDS,
1mgml 1 proteinase K) overnight, washed three times in TE buffer (10 mM
Tris-HCl, 1 mM EDTA, pH 8), and stored in TE at 4°C.
For PFGE analysis, 20 ng DNA or 5 10 7 phage particles were loaded in each
well. PFGE was performed on a CHEF-DR III system (Bio-Rad) using 1%
PFGE certified agarose (Sigma). The gel was run for 11 h in 0.5 TBE buffer
(1 TBE is 89 M Tris, 2 mM EDTA, and 89 mM boric acid, pH 8.3), at a 0.5-
to 5.0-s switch time, 6 V/cm, and an included angel of 120°. The gel was stained
with SYBR gold (Molecular Probes) for 30 min, destained in 0.5 TBE for 30
min, and photographed (Gel Doc XR; Bio-Rad). Genome sizes were determined

6732 HOLMFELDT ET AL. APPL. ENVIRON. MICROBIOL.
Taxon
TABLE 1. Genetic and morphological characteristics of bacterial strains used in this study
Strain
Isolation
yr
Isolation
location
homology b 16S rRNA gene ITS UP-PCR LPS
Colony
morphology
% Similarity c (GenBank accession no.) Group no. according to:
% DNA
Cellulophaga NN015840 a 1994 Svaneke 100 100.0 (AJ005972) 100.0 (EF667121) 10 1 7
baltica NN016038 1994 Svaneke 100 100.0 (EF667101) 98.7 (EF667122) 9 1 7
NN015839 1994 Svaneke 100 100.0 (EF667102) 99.1 (EF667123) 10 1 7
NN014845 1994 Svaneke 100 100.0 (EF667103) 99.6 (EF667124) 10 1 6
NN014847 1994 Svaneke 102 100.0 (EF667104) 98.7 (EF667125) 9 1 8
NN014850 1994 Svaneke 100 100.0 (EF667105) 99.9 (EF667126) 10 1 6
NN014873 1994 Svaneke 102 100.0 (EF667106) 99.9 (EF667127) 10 1 6
NN016046 1994 Ellekilde 78 100.0 (EF667107) 98.8 (EF667128) 2 1 10
Hage
NN016048 1994 Ellekilde 71 100.0 (EF667108) 98.1 (EF667129) 3 1 10
Hage
MM#3 2000 Øresund 100.0 (AF497997) 98.4 (EF667130) 2 1 2
#4 2000 Øresund 100.0 (EF667109) 98.5 (EF667131) 4 1 2
#10 2000 Øresund 100.0 (EF667110) 98.7 (EF667132) 5 1 7
#12 2000 Øresund 100.0 (EF667111) 98.5 (EF667133) 6 1 3
#13 2000 Øresund 99.5 (EF667112) 98.8 (EF667134) 1 1 9
#14 2000 Øresund 99.5 (EF667113) 98.5 (EF667135) 2 1 4
#17 2000 Øresund 99.5 (EF667114) 98.1 (EF667136) 7 1 4
#18 2000 Øresund 100.0 (EF667115) 99.0 (EF667137) 8 1 5
#19 2000 Øresund 99.5 (EF667116) 98.2 (EF667138) 2 1 4
OL12a 2005 Off Öland 99.5 (EF667117) 98.9 (EF667139) 10 1 8
#3 r S M Laboratory 100.0 (EF667118) 98.4 (EF667140) 2 1 1
#3 r S T Laboratory 100.0 (EF667119) 98.4 (EF667141) 2 1 2
Cellulophaga
fucicola
Flavobacteriaceae
bacterium IE-7
NN015860 1994 Hirsholm 0 92.3 (AJ005973) 68.4 (EF667142) 11 2 11
#8 2000 Øresund 88.2 (EF667120) 75.1 (EF667143) 12 1 12
a C. baltica type strain (23).
b DNA homology with NN015840 (23).
c Sequence similarity to NN015840.
manually using an 8- to 48-kb standard (Bio-Rad) and a PFGE marker (Amersham)
as molecular size markers.
Restriction digests of phage genomes. Some phages with identical genome
sizes showed differences in host range. To examine whether these phages were
genetically different, they were compared by RFLP analysis. HindIII was chosen
after testing four different restriction enzymes (NdeII, HaeIII, EcoRI, and
HindIII; Boehringer Mannheim), and phage DNA (100 ng) was cleaved according
to the manufacturer’s recommendation. The restriction digests were analyzed
on an agarose gel (0.7%, 90 V, 2 h), stained with ethidium bromide, and photographed
as described above. A GeneRuler 1-kb DNA ladder (Fermenta) was
used as a molecular size marker.
TEM. Selected phages representing a range in genome size and host range
were examined by transmission electron microscopy (TEM). Freshly made lysates
were obtained from fully lysed plates (see above). Grids were prepared by
placing 8 l of lysate onto 200-mesh Formvar-coated copper grids (Ted Pella) for
2 min. The phage solution was removed with filter paper, and grids were subsequently
stained with 8 l 2% sodium phosphotungstate (phages 40:2, 18:4,
18:4, 38:1, 39:1, 4:1, and 3:2) or 2% uranyl acetate (phages 13:1, 18:1,
12:1, S M , and 10:1) for 2 min. The grids were examined using a Zeiss EM
900 microscope with an accelerating voltage of 80 kV.
Nucleotide sequence accession numbers. The partial 16S rRNA gene sequences
and ITS sequences obtained in this study have been deposited in
GenBank and assigned accession numbers EF667101 to EF667143 (Table 1).
Bacterial characterization. A total of 23 bacterial strains
obtained from five localities (Fig. 1) in 1994, 2000, and 2005
were characterized and grouped according to the parameters
shown in Table 1. The C. baltica strains formed yellow colonies
on nutrient agar plates and could be divided into 10 morphological
groups. Only four of the C. baltica strains were regarded
as having unique colony morphology. The remaining isolates
were identical to up to three other isolates. For the phageresistant
strains, strain #3 r S T had morphology identical to
that of MM#3, while strain #3 r S M differed (Table 1).
RESULTS
FIG. 1. Sampling sites for bacterial (b) and phage (p) isolations.
Exact locations are marked by arrows. b1, Hirsholm; b2, Ellekilde
Hage; b3, Svaneke; b4, Øresund; b5, off Öland. The map was generated
using the Online Map Creation software (http://www.aquarius
.geomar.de/omc/).

VOL. 73, 2007 COMPLEXITY OF FLAVOBACTERIUM PHAGE-HOST INTERACTIONS 6733
FIG. 2. Neighbor-joining trees showing the phylogenetic relationships between bacteria used in this study. Trees are based on 16S rRNA gene
sequences (800 bp) (A) and ITS gene sequences (675 bp) (B). Phylogenetic relationships were bootstrapped 1,000 times, and values greater
than 50% are shown.
Sequencing of the 16S rRNA gene and ITS. Partial sequencing
of the 16S rRNA gene (800 bp) showed that 15 strains
were identical to the C. baltica type strain NN015840, while 5
strains showed a 4-bp difference from this bacterium (Table 1;
Fig. 2A). Strains NN015860 (Cellulophaga fucicola, accession
no. AJ005973) and #8 (98% similar to Flavobacteriaceae bacterium
IE-7, accession no. EF105392) showed only 93% and
88% 16S rRNA gene similarity to the other strains (Fig. 2A).
These strains also had shorter ITS (800 and 700 bp, respectively)
than the C. baltica strains (900 bp). All the C. baltica
strains showed high ITS sequence similarity (98 to 100%) (Table
1) and formed a tight phylogenetic cluster (Fig. 2B).
UP-PCR and LPS profiling. Due to the high sequence similarities
of 16S rRNA genes and ITS, we sought to distinguish
the C. baltica strains by means of UP-PCR fingerprinting. Accordingly,
the C. baltica strains could be divided into 10 different
UP-PCR groups (Table 1). Still, several of the strains
had identical UP-PCR fingerprints, and three of the UP-PCR
groups contained two to six identical isolates (Fig. 3).
Acquisition of phage resistance may yield detectable
changes in LPS profiles (see, e.g., references 18, 37, and 56);
however, we observed no differences in LPS profiles among
the C. baltica isolates despite their pronounced variation in
phage susceptibility (see below). Only isolate NN015860 (C.
fucicola) had a different LPS profile (Table 1; see Fig. S1 in
the supplemental material).
Phage characterization. Phages were obtained from Øresund
in 2000 and 2005 using plaque assays (Fig. 1). Plaques
were isolated for all bacterial host strains except for strains #3
r S M , #8, and NN014845, and a total of 45 phages infecting
the C. baltica strains and 1 phage infecting C. fucicola were
obtained. From a single water sample, up to four different

6734 HOLMFELDT ET AL. APPL. ENVIRON. MICROBIOL.
FIG. 3. Genomic profiling of bacterial isolates. An agarose gel
showing UP-PCR banding patterns for 15 of the 23 bacterial isolates
analyzed is shown. Identical strains (e.g., strains NN015839 and
NN015840) and strains with differences (e.g., strains 10 and 12) are
displayed.
plaque morphologies could be distinguished for a single bacterial
host (e.g., 48:1 to 48:4 [Table 2]).
Phage genome size and morphology. To genetically distinguish
the phages, genome sizes were determined by PFGE
analysis. Phage genome sizes ranged from 8 to 242 kb and
represented 18 different sizes (Table 2; Fig. 4A). Several
phages had identical genome sizes, and only 10 phages were
unique in size (Table 2). Some phages showed double bands on
the PFGE with up to a 4-fold difference in molecular size.
For example 3:2 had bands of 18 kb and 76 kb. Some phages
with very different genome sizes were infectious to the same
bacterial strain. For instance, strain NN015839 could be infected
by both 40:2 ( 242 kb) and 39:1 (29 kb).
Selected phages showing a large range in genome size (8 to
242 kb) and host range (1 to 20 hosts) represented a wide
variety of morphotypes (Fig. 5) and belonged to the Myoviridae,
Siphoviridae, and Podoviridae families (1) (Table 2).
Broad- and narrow-host-range phages were found in all three
families. Two phages showing two bands each in the PFGE
analysis were examined by TEM. For phages 3:2 and 18:4,
one and two morphotypes could be observed, respectively (Fig.
5). Interestingly, some phages with identical genome sizes, e.g.,
12:1 and 18:1, belonged to different families (Table 2;
Fig. 5).
Host range and diversity of obtained phages. To examine
whether the phages were specific for C. baltica, infectivity was
tested on five Bacteroidetes isolates, one alphaproteobacterium,
and one gammaproteobacterium. No infections were
observed (data not shown). Also, no cross-infectivity was observed
for C. baltica and C. fucicola phages, and no phages
infected strain #8 (Fig. 6). Within C. baltica, the phages
showed a large variation in infectivity (Fig. 6). Six phages
showed narrow host specificity, infecting only the bacterial
strain with which they were originally isolated, but a broader
host range was more common. A few phages were able to
infect all bacterial strains except strains #3 r S M and #3 r
S T . No relationship between host range and time of isolation
was found.
In some cases there were no or only small differences in host
range between phages with identical genome size. To determine
whether these phages were genetically different, they
were examined by RFLP analysis. The RFLP patterns showed
that phages with even minor differences in host range were
genetically different and therefore should be regarded as
unique (Fig. 4B). For instance, phages 4:1 and 17:2 had a
genome size of 145 kb (Table 2), differed with respect to
infection of a single bacterial strain (Fig. 6), and showed
slightly different RFLP profiles (Fig. 4B). Phages with identical
genome sizes, host ranges, and RFLP patterns were considered
identical (e.g., phages 12:1 and 12:3 to 12:5 [Table 2; Fig.
4 and 6]). Of the 45 isolated phages, 40 were concluded to be
unique. In all cases, identical phages originated from the same
water sample.
Phage
TABLE 2. Phages isolated and analyzed in this study
Original
host strain
Isolation
yr
Genome size
(kb) a
Phage family b
40:1 NN015840 2005 68 ND
40:2 NN015840 2005 242 Myoviridae
38:1 NN016038 2005 70 Podoviridae
38:2 NN016038 2005 54 ND
39:1 NN015839 2000 29 Siphoviridae
39:2 NN015839 2005 242 ND
47:1 NN014847 2005 54 ND
50:1 NN014850 2005 242 ND
73:1 NN014873 2005 242 ND
46:1 NN016046 2000 38 ND
46:2 NN016046 2005 21/85 ND
46:3 NN016046 2005 78 ND
46:4 NN016046 2005 145 ND
48:1 NN016048 2005 9/19 ND
48:2 NN016048 2005 18/72 ND
48:3 NN016048 2005 18/72 ND
48:4 NN016048 2005 145 ND
S M MM#3 2000 54 Myoviridae
S T MM#3 2000 97 ND
3:1 MM#3 2000 54 ND
3:2 MM#3 2005 18/76 Myoviridae
4:1 #4 2005 145 Myoviridae?
10:1 #10 2000 54 Myoviridae
12:1 #12 2000 40 Siphoviridae
12:2 #12 2005 8/17 ND
12:3 #12 2000 40 ND
12:4 #12 2000 40 ND
12:5 #12 2000 40 ND
13:1 #13 2000 97 Siphoviridae
13:2 #13 2005 78 ND
14:1 #14 2005 9/19 ND
14:2 #14 2005 110 ND
17:1 #17 2000 42 ND
17:2 #17 2005 145 ND
18:1 #18 2000 40 Podoviridae?
18:2 #18 2000 40 ND
18:3 #18 2005 78 ND
18:4 #18 2005 9/19 Siphoviridae/
Siphoviridae
19:1 #19 2000 60 ND
19:2 #19 2000 97 ND
19:3 #19 2005 70/80 ND
19:4 #19 2005 242 ND
12a:1 OL12a 2005 9/19 ND
3S T :1 #3 r S T 2005 18/76 ND
3S T :2 #3 r S T 2005 54 ND
60:1 NN015860 2000 40 ND
a The presence of two values indicates two bands in the PFGE.
b ND, no data.

VOL. 73, 2007 COMPLEXITY OF FLAVOBACTERIUM PHAGE-HOST INTERACTIONS 6735
FIG. 5. TEM of selected C. baltica phages. Bars, 50 nm. (A) 40:2;
(B) 18:4; (C) 18:4; (D) 38:1; (E) 13:1; (F) 39:1; (G) 4:1;
(H) 3:2; (I) 18:1; (J) 12:1; (K) S M ; (L) 10:1. The phage 18:4
lysate included two morphotypes (B and C). Examples of phages with
identical genome sizes but with different morphotypes are shown (e.g.,
I versus J and K versus L). Genome sizes and host ranges for the
phages shown are given in Table 2 and Fig. 6.
FIG. 4. Genomic characterization of phages. (A) PFGE analysis of
genomic DNAs from 9 of the 46 phage isolates used in this study. The gel
displays phages with identical (e.g., 12:4 and 12:5) and different (e.g.,
18:3 and 39:1) genomes sizes. (B) HindIII restriction digests of eight
phage genomes. Cleavage patterns for phages with identical (12:4 and
12:5) and different (e.g., 17:1 and 18:1) infectivity patterns are shown
along with patterns for phages with almost identical infectivity patterns
(4:1 and 17:2; see Fig. 6). Arrowheads indicate differences in banding
patterns for 4:1 and 17:2. See Table 2 for phage genome sizes.
Susceptibility and resistance to phage infection. The phage
susceptibility of the C. baltica strains was highly variable. For
instance, the most (strain #13) and the least (strain NN014845)
susceptible strains could be infected by 27 and 12 different phages,
respectively (Fig. 6). All strains showed unique susceptibility patterns.
To facilitate a comparison with other bacterial characteristics,
the phage susceptibility data were analyzed by
UPGMA and converted to a dendrogram (Fig. 7). In some
cases the clustering was consistent with genetic or morphological
characteristics. For instance, for strains NN015840,
NN014845, NN014850, and NN014873, the phage susceptibility
was consistent with high similarity of the 16S rRNA
gene, UP-PCR, and colony morphology as well as with a
100% match in DNA homology (23). Strains #13, #14, #17,
and #19 grouped according to 16S rRNA gene sequences
and phage susceptibility but showed small differences in
UP-PCR pattern and colony morphology (Fig. 2A and 7).
For most strains, no consistency was observed between
phage susceptibility and any of the analyzed strain characteristics.
The effects of resistance acquisition were examined for
strain MM#3. When resistance against S M was acquired,
MM#3 became resistant towards 21 phages, while resistance
against S T conferred resistance against only 3 phages (Fig. 6).
To determine whether phage infection was equally efficient in
different hosts, infectivity was examined for three phages by exposing
three susceptible hosts to the same phage titer (Table 3).
The number of PFU varied up to 6 orders of magnitude between
host strains.
DISCUSSION
Similarity of the bacterial strains. C. baltica strains are
members of the Flavobacteria class within the Bacteroidetes,
which is one of the dominant phyla in marine waters (16, 25).
Flavobacteria in particular are thought to be important during
phytoplankton blooms (42, 46). Therefore, the ecology of the
C. baltica-phage system could be an important part of carbon
cycling associated with phytoplankton blooms. According to
16S rRNA gene sequences, the C. baltica strains were closely
related. Therefore, sequencing of the ITS was performed to
differentiate the strains. The ITS is known to differ in length
and/or nucleotide composition within species (47, 49), and
recently it was shown that bacterial groups showing 99% 16S
rRNA gene similarity may have ITS sequences with 93%
identity (8). Still, the C. baltica strains showed 98% ITS
sequence similarity, confirming that there is no overall relationship
between 16S rRNA gene and ITS sequence similarity
(8). Since the different strains were not widely distinguishable
by 16S rRNA gene and ITS sequencing, a whole-genome PCR
typing analysis (UP-PCR) was performed. Indeed, using this
method the strains could be divided into 10 groups based on
banding patterns, but three groups still contained two to six
isolates. Combined with an earlier analysis showing 100% hybridization-based
DNA-homology for a number of the strains
(Table 1) (23), our molecular characterizations show that the
C. baltica strains are highly homologous at the genomic level.
Therefore, these bacterial strains were considered to be well
suited for this study of phage-host interactions.
Phage diversity and host range. The C. baltica phages included
representatives of Myoviridae, Siphoviridae, and
Podoviridae and ranged in genome size from 8 to 242 kb. This
is a significantly wider range than for other isolated aquatic
phages (30 to 125 kb) (12, 51, 67) or for marine phage
communities (25 to 70 kb), where genomes of 100 kb often

6736 HOLMFELDT ET AL. APPL. ENVIRON. MICROBIOL.
FIG. 6. Host ranges of the 46 phages. Gray squares indicate lysis and white squares indicate no lysis.
are assumed to originate from algal viruses (54, 68). Nine of
the 45 C. baltica phages isolated in the present study were
100 kb, and recently a phage genome of 200 kb from
polluted seawater was characterized (36). Hence, obviously
large genome viruses in natural assemblages cannot be assumed
to be phytoplankton viruses.
In some cases the PFGE analysis yielded double bands for a
phage stock. This phenomenon has previously been recognized
as multiple-phage infection according to electron microscopy
(12). Accordingly, two phage morphotypes were observed for
18:4. However, for 3:2 only one morphotype was detected.
It remains unclear whether a second phage was present at low
abundance in the 3:2 lysate and therefore was not detected or
whether the two bands originate from a single phage with a
fragmented genome (see, e.g., reference 33). Of the 45 C.
baltica phages, 40 were unique according to genome size and
host range. Selected phages with identical genome sizes were
found to belong to different families according to TEM analysis.
Further, RFLP digests demonstrated that even phages
that differed only with respect to infection of a single host
strain could be distinguished. Hence, along with other recent
studies (2, 12), our data highlight that whole-genome finger-

VOL. 73, 2007 COMPLEXITY OF FLAVOBACTERIUM PHAGE-HOST INTERACTIONS 6737
FIG. 7. UPGMA tree based on susceptibility to phages infection. The data shown in Fig. 6 were scored and converted to pairwise distances
using the Dice similarity coefficient. Groupings of bacterial strains according to UP-PCR and LPS profiling and colony morphology are inserted
to facilitate comparison.
printing analyses of viruses with similar genome sizes may
reveal an otherwise concealed genetic and morphological variation.
This indicates that analyses of natural viral assemblages
based on genome size (i.e., PFGE) severely underestimate
genetic diversity and viral community dynamics.
Infection by the C. baltica phages appeared to be species
specific, with a wide intraspecies host range (Fig. 6) and a
tremendous variation in infectivity of specific hosts (Table 3).
This illustrates how phage community composition will have a
large impact on the way that phages influence mortality as well
as the species and strain compositions of natural bacterioplankton
communities. Further, since phages with up to 10-
fold differences in genome size were infectious to the same
host, it is possible that the extent and/or efficiency of transduction
events (41) and phage conversion (9) in the environment
will be highly dependent on infection by specific phages.
Strain-specific susceptibility and resistance to phage infection.
While the C. baltica strains appeared to be genetically
similar, they all showed a unique combination of susceptibility
TABLE 3. Efficiency of phage infection in three bacterial strains
Infection efficiency a in strain:
Phage
MM#3 #4 #10
S T 100 14 0.001
38:1 100 0.2 0.003
40:1 100 1 0.0008
a Strains were exposed to the same phage titer (10 8 PFU). Efficiency is
expressed in relative PFU, where the highest PFU was set to 100%.
to the tested phages. This was seen not only as the number of
phages infective to a given bacterial strain but also in relative
efficiency of infection when different hosts were exposed to the
same phage and titer (Table 3). Similarly, variable infectivity
has been observed for phages infecting different bacterial species
(22). Therefore, the impact of phage infection and lysis on
a bacterial population is dependent not only on whether the
phage can infect the host but, just as importantly, on how
susceptible the host is to infection. In E. coli, acquisition of
resistance, often associated with modification or loss of receptor
molecules to which the phage initially binds (5, 28, 43), may
occur in one or two steps leading to the presence of partially
resistant mutants (29). Hence, the specific mutation and receptor
in question determine the extent of resistance. This
complexity is also illustrated by acquisition of resistance in the
C. baltica strain MM#3. While resistance against S M conferred
resistance against a large number of phages, resistance
against S T gave resistance against only three phages with
similar genome size (Table 2; Fig. 6). This suggests that different
receptors are used by S M and S T .
In E. coli the acquisition of resistance is often coupled to
changes in the LPS structure, which are sometimes detectable
by SDS-polyacrylamide gel electrophoresis (37, 56). Using this
method, we found no differences between C. baltica strains,
despite the fact that all showed unique susceptibility patterns,
or between strain 8 and the C. baltica strains. This calls the
sensitivity of the method into question; however, it cannot be
excluded that susceptibility was determined by cellular features
other than LPS (e.g., cell surface proteins) (29).

6738 HOLMFELDT ET AL. APPL. ENVIRON. MICROBIOL.
Temporal and spatial co-occurrence of phage-host systems.
In the present study, hosts were isolated from five different
locations during a 10-year period, while phages were obtained
from a single location on two occasions with 5 years in between
(Fig. 1; Tables 1 and 2). The isolated assemblages consisted of
bacterial strains with variable susceptibility and phages with
large host range diversity. Comparable results have been obtained
for phage-host systems in other coastal regions. For
instance, in several successive years, Wichels et al. (64) isolated
Pseudoalteromonas phage-host systems from locations in the
German Bight and observed complex interactions between
phages and hosts at the strain level. Also, Comeau et al. (12)
showed that the vibriophage community of the Strait of
Georgia (Canada) consisted of a highly diverse mixture of
phenotypes and genotypes.
We obtained phages infectious to bacterial strains that were
isolated from localities hundreds of kilometers apart (e.g., see
locations p, b2, and b5 in Fig. 1) encompassing salinities from
7to18 practical salinity units. Interestingly, despite the fact
that one C. baltica strain was isolated from the Baltic Sea
(location b5 in Fig. 1), we were not able to isolate phages
infective to any of the C. baltica strains from this brackish
environment (data not shown). Hence, as also indicated by the
study by Wichels and coauthors (64), salinity may function as a
natural barrier for the distribution of some phage-host systems.
In oceanic waters, genetically related phages may be distributed
over thousands of kilometers (24).
Major findings and implications. This study shows that there
is a large variation in phage susceptibility within a well-defined
phylogenetic group of marine bacteria, the C. baltica strains, as
well as large host range diversity among C. baltica phages. Despite
the fact that only some of the 21 C. baltica strains were distinguishable
through genomic/membrane profiling and DNA sequencing,
they all showed unique phage susceptibility patterns,
thus indicating an extremely complex web of phage-host interactions
even within this restricted group of marine bacteria.
If our data on C. baltica-phage interactions are representative
of marine bacteria in general, the results add completely
new perspectives to the role of phages in bacterial diversity and
function. One potential implication is that phage-host interactions
may have gone undetected in studies concerning natural
communities of phages and bacteria (see, e.g., references 44
and 50) if they were based on the phylogenetic resolution
provided by the 16S rRNA gene or ITS. However, a few studies
based on 16S rRNA gene methodology have been able to
demonstrate viral control of single operational taxonomic units
(terminal RFLP) (66) or bacterial classes (fluorescent in situ
hybridization) (6). The observed variations in susceptibility
among our strains and in infectivity of individual phages predict
that the specific strain-level composition of co-occurring
bacterial and phage assemblages determines to what extent
infection and lysis affect bacterial community composition as
analyzed by 16S rRNA gene- or ITS-based methods. Conceivably,
the large diversity at the strain level implies that strains
exposed to phages are replaced by strains that are less susceptible
to the co-occurring phages and, consequently, that phage
activity is a driving force in the strain-specific diversity and
continuous succession in bacterial populations. Indeed, we
found that susceptible bacterial strains may differ by up to 6
orders of magnitude in sensitivity to the same titer of phage.
Hence, we speculate that marine bacterioplankton species are
susceptible to multiple co-occurring phages but that the sensitivity
to phage infection exists as a continuum between two
extremes, highly sensitive and resistant. The data presented
contribute to our emerging view of phage-host interactions in
marine waters but also illustrate that the link between phage
action and bacterial diversification is complex and poorly understood.
Future studies of basic phage-host interactions and
dynamics in their natural environment are, therefore, fundamental
for improving existing conceptual models on the role of
phages in bacterial diversity and population dynamics.
ACKNOWLEDGMENTS
We thank J. E. Johansen and P. Nielsen for providing strains
NN014847 to NN016048, B. Olsen for use of PFGE equipment, and S.
Iversen for excellent technical assistance with LPS and UP-PCR analyses.
We also thank two anonymous reviewers for thoughtful comments
on the manuscript.
This study was supported by Kalmar University (L.R.), the Danish
Natural Sciences Research Council and the Directorate for Food Fisheries
and Agribusiness (M.M.), and the Faculty of Life Sciences, University
of Copenhagen (O.N.).
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